Sustained H(2) production driven by photosynthetic water splitting in a unicellular cyanobacterium

Abstract

The relationship between dinitrogenase-driven H(2) production and oxygenic photosynthesis was investigated in a unicellular cyanobacterium, Cyanothece sp. ATCC 51142, using a novel custom-built photobioreactor equipped with advanced process control. Continuously illuminated nitrogen-deprived cells evolved H(2) at rates up to 400 µmol ⋅ mg Chl(-1) ⋅ h(-1) in parallel with uninterrupted photosynthetic O(2) production. Notably, sustained coproduction of H(2) and O(2) occurred over 100 h in the presence of CO(2), with both gases displaying inverse oscillations which eventually dampened toward stable rates of 125 and 90 µmol ⋅ mg Chl(-1) ⋅ h(-1), respectively. Oscillations were not observed when CO(2) was omitted, and instead H(2) and O(2) evolution rates were positively correlated. The sustainability of the process was further supported by stable chlorophyll content, maintenance of baseline protein and carbohydrate levels, and an enhanced capacity for linear electron transport as measured by chlorophyll fluorescence throughout the experiment. In situ light saturation analyses of H(2) production displayed a strong dose dependence and lack of O(2) inhibition. Inactivation of photosystem II had substantial long-term effects but did not affect short-term H(2) production, indicating that the process is also supported by photosystem I activity and oxidation of endogenous glycogen. However, mass balance calculations suggest that carbohydrate consumption in the light may, at best, account for no more than 50% of the reductant required for the corresponding H(2) production over that period. Collectively, our results demonstrate that uninterrupted H(2) production in unicellular cyanobacteria can be fueled by water photolysis without the detrimental effects of O(2) and have important implications for sustainable production of biofuels.

Importance: The study provides an important insight into the photophysiology of light-driven H(2) production by the nitrogen-fixing cyanobacterium Cyanothece sp. strain ATCC 51142. This work is also of significance for biotechnology, supporting the feasibility of "direct biophotolysis." The sustainability of the process, highlighted by prolonged gas evolution with no clear sign of significant decay or apparent photodamage, provides a foundation for the future development of an effective, renewable, and economically efficient bio-H(2) production process.

FIG 1

Gas dynamics by N-deprived Cyanothece 51142 cultures. Fine-scale resolution of production rate dynamics was obtained in the LED photobioreactor upon interruption of NH₄⁺ influx, during continuous sparging with 1.3% CO₂-Ar (a) or pure Ar (b).

FIG 2

Carbohydrate (a, b) and protein dynamics (c, d) during H₂ production within the LED photobioreactor, during continuous sparging with 1.3% CO₂-Ar (a, c) or pure Ar (b, d). Carbohydrate measurements are based on glucose equivalents; open circles denote glycogen determination. Protein measurements are standardized to bovine serum albumin. Average values and standard deviations are based on at least two independently assayed sample replicates at each time point.

FIG 3

Light saturation of O₂ and H₂ production performed in situ within the photobioreactor. Experiments were performed after 16 h of CO₂-deprived H₂ production. Values represent the averages from two individual curves, performed 3 h apart.

FIG 4

Effects of electron transport inhibitors in illuminated sealed tubes with an Ar atmosphere. (a) Incubation with DCMU for 72 h, using 10 ml chemostat cells. Control value (100%) was 3.28 mmol H₂ ⋅ mg Chl⁻¹. (b) Treatment with p-benzoquinone (BQ) for 1 h, at 170 µM. Cells were sampled from a CO₂-deprived bioreactor, 78 h after NH₄⁺ deprivation. Control values (100%, no BQ) were 34 and 346 µmol ⋅ mg Chl⁻¹ ⋅ h⁻¹ for H₂ and O₂, respectively.

FIG 5

F_v/F_m and ETR_max during H₂ production for CO₂-supplied (a) and CO₂-deprived (b) reactor, as determined with PAM fluorometry.